Depth Filtration as an Improved Method of Enzyme Removal in Biocatalytic Reactions

Biocatalytic transformations in small-molecule pharmaceuticals have unlocked the door to greener chemistries, simplified synthetic routes, and reduced the transition metal catalyst usage to deliver high-purity intermediates and active pharmaceutical ingredients. These benefits rely on the scalable removal of the enzyme for efficient product isolation. This study evaluates three enzyme removal methodologies employed during the belzutifan hydroxylation step synthesis: waste cake filtration using Celite 545 as a filter aid, liquid–liquid extraction (LLE), and direct filtration via depth filtration. Celite 545 waste cake filtrations were successful in removing the precipitated enzyme over a pH range of 3–5 and a temperature range of 40–55 °C; however, it required 2 kg Celite 545/kg of substrate loading to achieve reasonable filtration cycle times. LLE demonstrated potential for efficient enzyme removal, although challenges with phase separation and rag layer formation were encountered. Depth filtration, using ZetaPlus 05SP filters, proved effective at both lab and pilot scales, achieving enzyme loading capacities of ∼180 and ∼250 kg batch/m2, respectively, at 25 psi. For the belzutifan fluorination/DKR step, ZetaPlus R55SP-activated carbon filters were used to remove both the KRED enzyme and oligomer impurities simultaneously, achieving an ∼185 kg batch/m2 loading capacity in the pilot plant. The findings underscore the importance of optimizing enzyme removal strategies and highlight the need for continued innovation in small-molecule biocatalytic processes.